The respiratory epithelium of mammals is a complex tissue responsible for numerous physiological functions, one of which is forming a key barrier to potentially harmful environmental threats. Multiple defense mechanisms have been identified which protect the respiratory tract from inhaled agents that are known to be responsible for airway disease, such as infectious agents, gases, and particulates. Newhouse, M.T. and J. Bienenstock, "Respiratory Tract Defense Mechanisms," Textbook of Pulmonary Disease, Little, Brown and Comp (1989). These multiple defenses are the result of a combination of anatomical design of the airway, together with the physiological role of local and circulating cells.
Recent isolation and characterization of antimicrobial peptides in a variety of species and tissues has unveiled a new component of animal host defense. These various peptides, which can be classified into families based on common sequences, secondary structure and/or sites of activity, are believed to participate in defense against potential microbiological pathogens. Cecropins were the first well characterized family of structurally related antimicrobial peptides and are found in a wide distribution of insects. Boman, H.G. and D. Hultmark, Ann. Rev. Microbiol., 41:103-126 (1987). They are coordinately expressed in the fat body of insect larvae following infection or injury. In vertebrates, the magainin family of antimicrobial peptides have been isolated from glands of the skin and gastrointestinal tract of Xenopus Iaevis, and are thought to form the basis for a defense system of the amphibian mucosal surfaces against infection. Soravia, E., G. Martini et al., "Antimicrobial properties of peptides from Xenopus granular gland secretions," FEBS Lett., 228:337-40 (1988); Zasloff, M.A., "Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active froma, and partial cDNA sequence of a precursor," Proc Natl Acad Sci USA, 84:5449-53 (1987). Defensins are peptides found in phagocytic cells isolated from several mammalian species including man, and may be characterized by 8 invariant residues within the sequence. Gabay, J.E., "Microbicidal mechanisms of phagocytes," Curr Opin Immunol, 1(1):36-40 (1988); Gabay, J.E., R.W. Scott et al., "Antibiotic proteins of human polymorphonuclear leukocytes," Proc Natl Acad Sci USA, 86(14):5610-4 (1989); Ganz, T., "Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes," Infect Immun, 55(3):568-71 (1987): Ganz, T., J.A. Metcalf et al., "Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi syndrome and `specific` granule deficiency," J CIin Invest, 82(2):552-6 (1988); Ganz, T., J.R. Rayner et al., "The structure of the rabbit macrophage defensin genes and their organ-specific expression," J Immunol, 143(4):1358-65 (1989); Ganz, T., M.E. Selsted et al., "Antimicrobial activity of phagocyte granule proteins," Semin Respir Infect, 1(2):107-17 (1986); Ganz, T., M.E. Selsted et al., "Defensins," Eur J Hematol, 44(1):1-8 (1990a); Ganz, T., M.E. Selsted et al., "Defensins," Eur J Haematol, 44(1):1-8 (1990b); Ganz, T., M.E. Selsted et al., "Defensins. Natural peptide antibodies of human neutrophils," J Clin Invest, 76(4):1427-35 (1985). They possess antimicrobial activity in vitro against bacteria, fungi, and viruses, and may contribute to the "oxygen-independent" defense pathways of these cells. Lehrer, R.I., T. Ganz et al., "Oxygen-independent bactericidal systems. Mechanisms and disorders," Hematol Oncol Clin North Am, 2(1):159-69 (1988). Expression of defensin in a non-myeloid tissue source, the mouse small intestinal crypt cells, has also been reported. Ouellette, A.J., R.M. Greco et al., "Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium," J Cell Biol, 108(5):1687-95 (1989).
Cecropins, magainins, and defensins all share the properties of being cationic and membrane active, and evidence suggests that their antimicrobial activity is secondary to their ability to selectively disrupt membranes, possibly by channel formation. Bevins, C.L. and M.A. Zasloff, "Peptides from frog skin," Ann. Rev. Biochem., 59:395-414 (1990); Kagan, B.L., M.E. Selsted et al., "Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes," Proc Natl Acad Sci USA, 87(1):210-4 (1990); Lehrer, R.I., A. Barton et al., "Interaction of human defensins with Escherichia coli. Mechanism of bactericidial activity," J Clin Invest, 84(2):553-61 (1989); Zasloff, M.A., "Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active froma, and partial cDNA sequence of a precursor," Proc Natl. Acad Sci USA, 84:5449-53 (1987).
These newly emerging family of basic, cysteine-rich peptides with antimicrobial activity found throughout the animal kingdom include the defensins (Ganz, T., M.E. Selsted et al , "Defensins," Eur J Haematol, 44(1): 1-8 (1990b)), insect defensins (Lambert, J., E. Keppi et al., "Insect immunity:isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides" [published erratum appears in Proc Natl Acad Sci USA May;86(9):3321(1989)]. Proc Natl Acad Sci USA 86(1): 262-6 (1989)), bactenecins (Romeo D., B. Skerlavaj et al., "Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils," J Biol Chem, 263:9573-75 (1988)), sapecins (Matsuyama, K. and Natori, S. "Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Saroophaga peregrina,"J biol Chem, 263:17112-16 (1988)) and royalisin (Fujiwara, S., J. Imai et al., " A potent antimicrobial protein in royal jelly," J Biol Chem, 265: 11333-37 (1990)). Defensins are basic peptides of 30 to 34 amino acids with 3 disulfide bonds. The known characterized defensins from both myeloid and non-myeloid tissues all have highly conserved amino acid residues within the family, including 6 invariant cysteines. Aside from a pair of cysteine residues near the carboxy-terminus of the tracheal antimicrobial peptide of this invention, no consensus or other residues are shared between these peptides. Furthermore, the 5' region of all known defensin cDNAs are strikingly conserved even across species, and no similarity with this consensus region is found in the tracheal antibiotic peptide's cDNA. Comparison with the other cysteine containing antimicrobial peptides shows no similarity.
Formal searches of the NRBF protein data base using a modification (IBI) of fastP, Lipmann, D.J. and W.R. Pearson, "Rapid and sensitive protein similarity searches,.revreaction. Science, 227:1435-41 (1985) and the Intelligenetics Search found no protein sequences that disclose the tracheal antimicrobial peptides of the instant invention. A nucleotide-based search of the GenBank data base using the University of Wisconsin Genetics analysis software, Devereux, J., P. Haeberli et al., "A comprehensive set of sequence analysis programs for the VAX," Nucl. Acids Res., 12:387-95 (1984), was similarly unrevealing.